101CRYOPRESERVATION OF RAT EPIDIDYMAL SPERM: COMPARISON OF TWO COOLING 
PROTOCOLS

The present study examined post-thaw motility, plasma membrane integrity and fertility of rat epididymal sperm cooled by two procedures to +5°C, and then cryopreserved by controlled-rate freezing. Wistar rats were used in the present study. In protocol-I (2001 Reproduction 122, 463), epididymides were collected from a mature male and placed in a plastic dish containing 2mL of freezing medium I [23% (v/v) egg yolk, 8% (w/v) lactose monohydrate and antibiotics]. The epididymides were dissected with scissors to release epididymal sperm. The semen was kept at 15°C for 30min and then held at 5°C for 30min. The cooling rate from 15°C to 5°C was 0.3°Cmin−1. The cooled semen was diluted with 2mL of freezing medium II [freezing medium I with 1.4% (v/v) Equex Stm (ES, Nova Chemical Sales, Inc., Scituate, MA, USA)]. Mixed semen was aspirated into 0.25-mL straws and exposed to liquid nitrogen (LN) vapor for 10min. The straws were then plunged into LN. In protocol-II, epididymides were collected from a mature male and placed in 4mL of freezing medium III [freezing medium I and 0.7% (v/v) ES]. The epididymides were dissected with scissors and held for 10min at room temperature to release epididymal sperm. The semen was loaded into 0.25-mL straws and kept at 15°C for 15min and then held at 5°C for 15min. The cooling rate from 15°C to 5°C was 0.7°Cmin−1. The cooled straws were then exposed to LN vapor for 10min and plunged into LN. Straws were thawed in a 37°C water bath for 10s. Thawed semen in a straw was diluted with 1mL of KRB medium with 0.4% (w/v) bovine serum albumin (BSA, fraction V, Sigma, Tokyo, Japan) at 37°C and then incubated at 37°C in 5% CO2 in humidified air. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37°C under a light microscopy at 100×. The sperm membrane integrity was determined using a commercial Live/Dead sperm viability kit (Molecular Probes, Inc., Eugene, OR, USA) which differentiates between cells with intact plasma membranes and those with damaged membranes by fluorescent staining patterns observed with a fluorescence microscope (Olympus, IX-71, Tokyo, Japan). Similar levels of sperm motility were observed immediately after thawing of sperm from both protocols. However, after 2h of incubation, the post-thaw motility of sperm frozen by protocol-II was significantly (P<0.01) higher than that of protocol-I. Sperm membrane integrity immediately after thawing was also higher for sperm frozen by protocol-II (22.1% v. 9.3%, P<0.01). Sperm frozen/thawed by protocol-II was inseminated into the top of the uterine horns of recipient females to confirm fertility. Two of three inseminated females became pregnant and gave birth to 5 pups. These results suggest that loading sperm into straws before cooling and subsequent slow cooling at 5°C to 0.7°Cmin−1 increases post-thaw survival of rat epididymal sperm.

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Kriopreservasi Semen Kambing Boer dengan Konsentrasi Pengencer Nira Aren dan Gliserol Berbeda

Jurnal Ilmu dan Teknologi Peternakan Tropis ◽

10.33772/jitro.v6i1.5422 ◽

2019 ◽

Vol 6 (1) ◽

pp. 1

Author(s):

Muhammad Riyadhi ◽

Anis Wahdi ◽

Muhammad Rizal

Keyword(s):

Sperm Motility ◽

Membrane Integrity ◽

Egg Yolk ◽

Boer Goat ◽

Palm Juice ◽

Sperm Membrane ◽

Live Sperm

ABSTRAK Penelitian bertujuan untuk mengetahui efektivitas nira aren sebagai pengencer alternatif dalam proses pembekuan (kriopreservasi) semen kambing boer.Kriopreservasi semen kambing boer menggunakan pengencer tris-gliserol-kuning telur (P1 73-7-20%), nira aren-gliseol-kuning telur(masing-masing P2 74-6-20%, P3 73-7-20%, dan P4 72-8-20%) dan andromed (P5 tanpa mengandung kuning telur dan gliserol). Parameter evaluasi meliputi motilitas, viabilitas, dan membrane plasma utuh setelah pengenceran, ekuilibrasi dan thawing. Evaluasi motilitas pasca thawing menunjukkan P5 52% berbeda nyata (P<0.05) dengan P1 42%, selanjutnya P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 8%, P3 6% dan P4 12%. Viabilitas pasca thawing menunjukkan P5 65,4% tidak berbeda nyata (P>0,05) dengan P1 61,8%, akan tetapi P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 26,2%, P3 29,8%, dan P4 34%. Membran plasma utuh (MPU) pasca thawing menunjukkan P5 66,2% tidak berbeda nyata (P>0,05) dengan P1 65,4%, akan tetapi keduanya berbeda sangat nyata (P<0.05) dengan P2 39%, P3 38%, dan P4 36,2%. Disimpulkan kriopreservasi semen kambing boer dengan pengencer nira aren dan gliserol pada konsentrasi berbeda belum dapat dipergunakan sebagai sumber bibit berdasarkan standar nasional Indonesia.Kata Kunci : Kambing boer, semen, nira arenABSTRACTThe experiment was conducted to determine the effect of sugar palm juice as alternative extender for cryopreservation process of boer semen.Tris-glycerol-egg yolk (P1 73-7-20%), Sugar palm juice-glyserol-egg yolk (P2 74-6-20%, P373-7-20%, dan P4 72-8-20%), and andromed (P5) used as a extender in the cryopreservation process of boer semen. Sperm motility (%), live sperm (%) and sperm membrane integrity (%) were recorded after diluted, equilibration and freeze-thawing. Result of post thawing motility showed that P5 52% was significantly different (P <0.05) with P1 42%, then P5 and P1 were significantly different (P <0.05) with P2 8%, P3 6% and P4 12%. Viability after thawing showed P5 65.4% was not significantly different (P> 0.05) with P1 61.8%, but P5 and P1 significantly different (P <0.05) with P2 26.2%, P3 29.8 %, and P4 34%. Spermmembrane integrity post-thawing showed P5 66.2% was not significantly different (P> 0.05) with P1 65.4%, but both were very significantly different (P <0.05) with P2 39%, P3 38% and P4 36.2%. Conclusions, sugar palm juice-glycerol-egg yolk with differentconcentrationsineffectively as an alternative extenderin cryopreservation of boer semen.Keywords: boer goat, semen, sugar palm juice

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Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c)

Zygote ◽

10.1017/s0967199418000230 ◽

2018 ◽

Vol 26 (4) ◽

pp. 301-307 ◽

Cited By ~ 2

Author(s):

José A. B. Bezerra ◽

Andréia M. Silva ◽

Patrícia C Sousa ◽

Lívia B. Campos ◽

Érica C. G. Praxedes ◽

...

Keyword(s):

Sperm Quality ◽

Semen Analysis ◽

Membrane Integrity ◽

Egg Yolk ◽

Coconut Water ◽

Computer Assisted ◽

Epididymal Sperm ◽

Sperm Plasma Membrane ◽

Pecari Tajacu ◽

Functional Membrane

SummaryThe aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

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54 CRYOPRESERVATION OF DOMESTIC CAT EPIDIDYMAL SPERM IN A DEFINED EXTENDER WITHOUT ANIMAL OR PLANT PROTEINS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab54 ◽

2012 ◽

Vol 24 (1) ◽

pp. 139

Author(s):

J. R. Saenz ◽

C. Dumas ◽

B. L. Dresser ◽

M. C. Gómez ◽

R. A. Godke ◽

...

Keyword(s):

Membrane Integrity ◽

Egg Yolk ◽

Domestic Cat ◽

The Other ◽

Initial Assessment ◽

Computer Assisted ◽

Plant Proteins ◽

Epididymal Sperm ◽

Sperm Suspension ◽

Sperm Survival

Previously, we have shown that survival of cat sperm is maintained in both non-egg yolk, semi-defined extenders and in extenders with greatly reduced levels of egg yolk (2%). Usually, cryoprotectant is added to extended samples after gradual cooling to 4°C, but recent reports have shown that satisfactory sperm survival can be obtained after addition at 22°C. Here, our objectives were to examine sperm survival after (1) cryopreservation from 22°C vs after gradually cooling to 4°C or (2) cryopreservation in a completely defined extender without animal or plant proteins vs extender + 2% egg yolk. Epididymides from local veterinary clinics were dissected in HEPES 199 medium (He199). The sperm suspension was filtered (40 μ), layered onto a density gradient column and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL of He199 and centrifuged for 5 min at 800 × g and the subsequent pellet was extended in TEST Buffer with either 0% (0% EY) or 2% egg yolk (2% EY). Next, 0% EY samples were further split into 2 groups—either gradually cooled to 4°C before 12% glycerol (1:1) was added (4C-0%EY) or 12% glycerol (1:1) was added at 22°C without cooling (22C-0%EY). Control samples extended in 2% EY were cooled to 4°C before addition of 12% glycerol (1:1) (4C-2%EY). Samples were loaded into 0.25-mL straws and placed in a –80°C freezer for 20 min before storage in LN2. Sperm samples were thawed in air (22°C) for 5 s and immersed in a 60°C water bath for 5 s. After a 7-step addition of He199, samples were centrifuged at 800 × g for 5 min and pellets resuspended in He199. Sperm samples were evaluated for motility (Mot; computer-assisted semen analysis, 37°C) at 0 h (initial assessment), after cooling to 4°C (PC) and at 0-h (0-PT) and 3-h post-thaw (3-PT) incubation at 37°C. Membrane integrity (MI; SYBR 14-PI) and acrosomal status (AS; FITC-PNA) were analysed at the initial assessment, 0-PT and 3-PT. Results are shown in Table 1. At 4°C (PC), sperm extended in 0% EY and 2% EY maintained 92 and 91%, respectively, of their initial motility (66%). At 0-PT and 3-PT, motility in the 3 groups had decreased by >50% and >70%, respectively. Motility at 3-PT in the 22C-0%EY treatment was less than the other 2 treatments (P < 0.05; 1-way ANOVA). At 0-PT, samples in the 4C-2%EY group had a higher membrane integrity value (P < 0.05) than did the 22C-0%EY group, whereas that of the 4C-0%EY group was not different from the other 2 groups. However, at 3-PT, both groups cooled to 4°C before cryopreservation had higher membrane integrity values (P < 0.05) than the group cryopreserved at 22°C. At 0-PT and 3-PT, the percentage of sperm with intact acrosomes ranged from 69% (4C-2%EY) to 59% (22C-0%EY) and from 55% (4C-2%EY) to 43% (22C-0%EY) of the initial value (89%), respectively. In summary, we demonstrated that cat epididymal sperm could be frozen successfully in a completely defined TEST-buffered extender. Furthermore, we confirmed that addition of cryoprotectant (i.e. glycerol) after gradual cooling to 4°C is beneficial to post-thaw survival. Table 1.Motility (Mot), membrane integrity (MI) and acrosomal status (AS) of cat epididymal sperm before and after cryostorage

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Slow freezing versus vitrification for the cryopreservation of zebrafish (Danio rerio) ovarian tissue

Scientific Reports ◽

10.1038/s41598-019-51696-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Lis S. Marques ◽

Ana A. N. Fossati ◽

Rômulo B. Rodrigues ◽

Helen T. Da Rosa ◽

Aryele P. Izaguirry ◽

...

Keyword(s):

Plasma Membranes ◽

Ovarian Tissue ◽

Membrane Integrity ◽

Egg Yolk ◽

Slow Freezing ◽

Mitochondrial Activity ◽

Trypan Blue Exclusion ◽

Cell Membrane Integrity ◽

Cell Plasma ◽

Cryopreservation Method

Abstract The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.

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10 Evaluation of Frozen Sperm Quality After Single Layer Centrifugation with Percoll Plus® of Fresh Bull Semen

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab10 ◽

2018 ◽

Vol 30 (1) ◽

pp. 144

Author(s):

F. N. Marqui ◽

A. Martins ◽

T. E. Cruz ◽

T. I. U. Berton ◽

C. P. Freitas-Dell'Aqua ◽

...

Keyword(s):

Artificial Insemination ◽

Sperm Quality ◽

Membrane Integrity ◽

Single Layer ◽

Annexin V ◽

Egg Yolk ◽

Molecular Probes ◽

Becton Dickinson ◽

Time Curve ◽

Intact Cells

The single layer centrifugation (SLC) with Percoll Plus® (PP; GE Healthcare, Uppsala, Sweden) before freezing is not a common technique used for selection of spermatozoa in bovine. Thus, this study aimed to verify the effect of SLC with PP before freezing on integrity of plasma and acrosome membranes (IPAM), phospholipid translocation (PT), and mitochondrial membrane potential (MMP) of frozen–thawed bull sperm. Three Nellore bulls housed at the Tairana Artificial Insemination Station were used. The ejaculates (6/bull) were collected by artificial vagina and assessed for sperm motility, concentration, and morphology. Then, the sperm were pooled and ~1 billion spermatozoa, either diluted [D; 1:2 (v/v)] in freezing extender (FE; tris, fructose, citric acid, egg yolk and antibiotics, without glycerol) or undiluted (UN), were placed on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90%, to form the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control (C), the supernatant was discarded and the pellet diluted in FE (plus glycerol) to a final concentration of 50 × 106 spermatozoa mL−1. Afterward, 0.5-mL straws were filled, cooled for 5 h at 4°C, and frozen in a programmable freezer (Digitcool, IMV, L’Aigle, France) following the temperature/time curve: from 4°C to –10°C (5°C min−1), –10°C to –100°C (40°C min−1) and from –100°C to –140°C (20°C min−1), in a total of 8 min, when the straws were plunged into and stored in liquid nitrogen until evaluation. Thawed sperm (at 37°C/30 s) was diluted at 5 × 106 spermatozoa mL−1 in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (100 μg mL−1; Sigma Co., St. Louis, MO, USA). After that, samples were stained for membrane integrity with the association of fluorescent probes propidium iodide (PI, 50 μg mL−1; Sigma Co.), fluorescein thiocyanate (FITC)-Pisum sativum agglutinin (PSA, 1 mg mL−1; Sigma Co.) and Annexin V-APC (BD Pharmingen, Franklin Lakes, NJ, USA), and with MitoStatus Red (20 nM; BD Pharmingen) and YO-PRO-1 (7.5 μM; Molecular Probes Inc., Eugene, OR, USA) for MMP. Sperm samples were analysed by flow cytometer (BD LSR; Fortessa, Becton Dickinson, Mountain View, CA, USA) and the results expressed as percentage of intact cells or qualitative fluorescence expressed in arbitrary units (AU). Analysis of variance and Tukey’s test were used for statistical analysis with P < 0.05 taken as significant. There were no differences between groups for IPAM (values ranging from 45.9 ± 7.0% to 55.6 ± 8.5%). Similarly, results of PT translocation did not differ among the groups (range from 34.7 ± 7.0% to 47.6 ± 7.0%). However, there was a tendency of increasing MMP (P = 0.10) in 70UD (1789 ± 258 UA), 70D (1776 ± 162.1 UA), and 90UD (1757 ± 133.8 UA) compared with C (1368 ± 267.4 UA) and 90D (1356 ± 145 UA). In conclusion, SLC did not compromise sperm membrane functionality and it seemed to select spermatozoa with higher mitochondrial functional activity when centrifuged at the concentration of 70% and 90D. This research was funded by FAPESP # 2015/20986-3, Tairana Artificial Insemination Station, MasterFertility Ltda, Brazil.

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255 PRONUCLEUS FORMATION IN RAT ZONA-FREE OOCYTES CO-CULTURED WITH HOMOLOGOUS POST-THAW SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab255 ◽

2005 ◽

Vol 17 (2) ◽

pp. 277

Author(s):

Y. Seita ◽

Y. Okuda ◽

A. Takizawa ◽

N. Hirahara ◽

M. Koichi ◽

...

Keyword(s):

Cooling Rate ◽

Sperm Motility ◽

Phase Contrast ◽

Room Temperature ◽

Membrane Integrity ◽

Egg Yolk ◽

Student’S T ◽

Percentage Data ◽

Student’S T Test

The aim of the present study was to develop an IVF system with frozen/thawed rat spermatozoa. We examined the effect of cooling rate to 5.0°C on post-thaw sperm motility and membrane integrity, and also investigated the ability of post-thaw spermatozoa to form pronuclei. Under room temperature, epididymal spermatozoa of Wistar rats were collected in 2.0 mL of egg yolk medium containing 8.0% (w/v) lactose monohydrate and 0.7% (v/v) Equex Stem. Samples were loaded into 0.25-mL straws and cooled to 5.0°C in the chamber of a programmed freezer. For cryopreservation, the samples were exposed to liquid nitrogen (LN) vapor for 10 min and then plunged into LN. Straws were thawed in a 37.0°C water bath for 10 s. Ovulated oocytes were collected and the zona pellucidae were removed with 0.1% pronase. One-hundred μL of thawed samples were put into a droplet of 400 μL R1ECM and pre-incubated for 1 h. R1ECM solution was added to the droplet to adjust to 0.5–1.5 × 106 sperm mL−1. The zona-free oocytes were then transferred into the droplet and co-cultured for 10 h. Oocytes were observed for pronuclei formation by means of an inverted phase contrast microscope. In Experiment I, the influence of sperm cooling rate to 5.0°C on sperm motility and membrane integrity was evaluated. Portions of samples were cooled at 54.0°C/min, 0.9°C/min, 0.5°C/min, and 0.3°C/min. The remainders were then frozen. The non-cooled samples were designated as controls. In Experiment II, we examined whether post-thaw spermatozoa have the ability to form pronuclei in vitro or not. All percentage data were arc-sine transformed and then analyzed by the Student's t-test. In Experiment I, the membrane integrity between the spermatozoa cooled at 0.5°C/min and the non-cooled spermatozoa was not different (38.1% vs. 37.2%; P > 0.05), but the integrity of these was higher than in spermatozoa cooled directly at 54.0°C/min (38.1% vs. 25.3%; P < 0.05). After culture for 1 h, the motility of spermatozoa cooled at 0.5°C/min was higher than that of those cooled at 54.0°C/min (61.3% vs. 53.3%; P < 0.05). At 2 h post-thaw the motility of spermatozoa cooled at 0.5°C/min was higher than that of spermatozoa cooled at 54.0°C/min and at 0.9°C/min (11.0% vs. 4.5%, 4.9%; P < 0.05). The membrane integrity of post-thaw spermatozoa cooled at 0.5°C/min was also higher compared to that of spermatozoa cooled at 54.0°C/min (22.5% vs. 8.4%; P < 0.01). In Experiment II, 28 (26.2%) of 107 oocytes had pronuclei when the post-thaw spermatozoa cooled at 0.5°C/min were used. The results indicated that the frozen/thawed spermatozoa cooled to 5.0°C at 0.5°C/min showed higher sperm motility and membrane integrity, and that spermatozoa can form pronuclei in homologous zona-free oocytes in vitro. Although in the rat sperm damage occurred during cooling to 5.0°C, and sperm motility and membrane integrity were also decreased by the cold shock, it is possible to decrease the damage by cooling slowly to 5.0°C at 0.5°C/min.

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89 SURVIVAL OF CAT EPIDIDYMAL SPERM AFTER TEMPORARY COOL STORAGE OR CRYOPRESERVATION IN DEFINED EXTENDERS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab89 ◽

2011 ◽

Vol 23 (1) ◽

pp. 150

Author(s):

J. R. Saenz ◽

C. Dumas ◽

B. L. Dresser ◽

M. C. Gómez ◽

R. A. Godke ◽

...

Keyword(s):

Membrane Integrity ◽

Egg Yolk ◽

Epididymal Sperm ◽

Sperm Analysis ◽

Santa Ana ◽

Cool Storage ◽

Sperm Suspension ◽

Sperm Survival ◽

Analysis System

A general objective of our studies on cat sperm is to enhance methods for both short- (+4°C) and long-term (–196°C) cryostorage, with particular focuses on improving compatibility with sex sorting and conforming to regulations for international shipment. Here, our specific aims were to a) determine the ability of cat sperm to survive during temporary cool storage in defined extenders (Exp. 1), and b) compare sperm survival after cryopreservation in the optimal defined extender v. TEST buffered extender + 2% egg yolk (TYB, Exp. 2). Testes from local veterinary clinics were transported in HEPES saline. Epididymides were dissected in HEPES 199 medium (He199), repeatedly sliced, and held at 37°C for ∼20 min. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL He199 and centrifuged for 5 min at 250 × g. In Exp. 1 (5 replicates), aliquots of the sperm pellet were extended in either of 2 defined extenders, Bioxcell® (BXC; IMV, Minneapolis, MN, USA) or HypoThermosol®-FSR (HTS; BioLife Solutions Inc., Bothell, WA, USA) or in TYB. Motility (Mot, Hamilton Thorne Sperm Analysis System CEROS 12, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at days 0, 1, 2, and 3 (Exp. 1), or after cooling (4°C) and post-thawing (p.t.), after 0 and 3 h incubation at 37°C (Exp. 2). In Exp. 2 (10 replicates), the sperm pellet was extended in BXC or TYB and gradually cooled to 4°C. Then, BXC or TYB + 12% glycerol was added (1:1) using a modified fixed osmolarity method (1995 Hum. Reprod. 10, 1109). Samples were loaded into 0.25-mL straws and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed in air (∼22°C) for 5 s and immersed in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps, centrifuged at 800 × g for 5 min, and pellets resuspended in He199. In Exp. 1, sperm in TYB, BXC, and HTS maintained 93, 69, and 56%, respectively, of initial motility (71%) after 3 days at 4°C (TYB > BXC and HTS; P < 0.05, 1-way ANOVA). Initially, 75 and 86% of sperm had membrane integrity and intact acrosomes, respectively. At 72 h, ∼80% of membrane intact sperm retained integrity in the two defined extenders v. nearly 90% in TYB (P > 0.05). At 24 h, all groups had high percentages of sperm with intact acrosomes (87 to 93%), but at 72 h, there was a difference between HTS (96%) and BXC (79%; P < 0.05). In Exp. 2 (Table 1), motility in TYB and BXC at 0 h p.t. was 77 and 70% of pre-freeze values – 77% (TYB) and 73% (BXC), respectively. Motility at 3 h p.t. was similar (BXC = 35% v. TYB = 37%). Membrane integrity and acrosomal status at 3 h p.t. ranged from 60% (BXC) to 72% (TYB) and from 65% (BXC) to 68% (TYB) of pre-freeze values, respectively. At 3 h p.t. M.I. of sperm in TYB was higher (P < 0.05) than in BXC. In summary, we have shown that cat epididymal sperm can be stored temporarily and cryopreserved successfully in a defined extender without animal proteins. Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

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70 REFREEZING POST-THAWED GOAT SEMEN

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab70 ◽

2011 ◽

Vol 23 (1) ◽

pp. 140

Author(s):

D. B. Carwell ◽

B. R. Scott ◽

G. T. Gentry ◽

K. R. Bondioli ◽

R. A. Godke

Keyword(s):

Liquid Nitrogen ◽

Sperm Morphology ◽

Membrane Integrity ◽

Egg Yolk ◽

Slow Cool ◽

Semen Parameters ◽

Slow Cooling ◽

Freeze Thaw ◽

Thaw Cycle ◽

Frozen Semen

The ability to successfully refreeze caprine sperm could provide a means of salvaging semen that was mistakenly thawed. The objective of this study was to compare treatment post-thaw semen parameters of twice-frozen caprine semen. Frozen semen from six mature Boer bucks (range in age from 2 to 6 years) was utilised for this experiment. Semen from each buck was extended in an egg yolk-based extender and packaged in 0.5-mL plastic straws before freezing and stored in liquid nitrogen. Three units of frozen semen from each buck was randomly allotted to each of four treatments as follows: (A) thaw and evaluate (control), (B) thaw, then plunge into liquid nitrogen, thaw, and evaluate, (C) thaw, incubate for 3 min at 37°C, slow cool and freeze, thaw, and evaluate, and (D) thaw, incubate for 5 min at 37°C, slow cool and freeze, thaw, and evaluate. Post-thaw parameters included total motility (TM), progressive motility (PM), membrane integrity (MI), and sperm abnormalities (AB). To obtain MI and AB, samples were stained with an eosin-nigrosin stain. A computerized programmable freezer was used to refreeze semen samples in treatment (Trt) C and Trt D. During the slow cooling portion of the protocol, samples were allowed to equilibrate at 38°C, then cooled to 4°C at a rate of 0.30°C min–1, and then held for 5 min. Samples were then cooled to –8°C at a rate of 15°C min–1, seeded, and cooled to –10°C at 15°C min–1, samples were then ramped to –80°C at 30°C min–1 before plunging into liquid nitrogen. Results indicate that post-thaw TM was significantly greater for Trt A (60%) when compared with Trt B, C, and D (0.05, 35, and 39%, respectively). Mean TM were not different between Trt C (35%) and Trt D (39%) but were greater than that for Trt B (0.05%). The PM for post-thaw semen in Trt A was also significantly greater (P < 0.05) when compared with that for Trt B and C (0.05 and 25%); however, no difference was found for mean PM for Trt A (47%) and Trt D (30%), nor were differences found between Trt C (25%) or Trt D (30%). Membrane integrity was higher in Trt A (27%) when compared to Trt B (2.2%). No differences in membrane integrity where found between Trt A, C, and D (27, 13, and 14%, respectively). Additionally, no differences were found between Trt B, C, and D for membrane integrity. Sperm morphology were not different were found with across all treatment groups. These results (i.e. Trt C and D) indicate that semen from mature Boer bucks can undergo a second freeze thaw cycle and still retain motility without dramatically affecting sperm morphology and membrane integrity. These findings indicate that directly plunging recently thawed semen back into liquid nitrogen should not be used for artificial insemination.

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Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants

Reproduction Fertility and Development ◽

10.1071/rd14449 ◽

2016 ◽

Vol 28 (12) ◽

pp. 1990 ◽

Cited By ~ 15

Author(s):

D. Acha ◽

M. Hidalgo ◽

I. Ortiz ◽

M. J. Gálvez ◽

J. J. Carrasco ◽

...

Keyword(s):

Sperm Motility ◽

Sperm Quality ◽

Membrane Integrity ◽

Egg Yolk ◽

Equus Asinus ◽

Sperm Membrane ◽

Freeze Test ◽

Pooled Samples

The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1% EG and 1.5% DMFA. Gent B yielded a higher (P < 0.01) post-thaw sperm motility than modified INRA96. EG 1% increased the sperm membrane integrity (P < 0.001), whereas DMSO affected sperm motility and membrane integrity (P < 0.001). DMFA 2.5% yielded higher (P < 0.001) values for sperm motility and membrane integrity. We concluded that Gent B improves in vitro post-thaw sperm quality of donkey spermatozoa, but the replacement of glycerol with 1% EG or 2.5% DMFA increased sperm protection against cryodamage. The use of DMSO for freezing donkey semen was unsuccessful and a toxic effect is suspected. These extenders should be included in the pre-freeze test for each donkey.

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Effect of Addition of Moringa Oleifera Extract to Tris Extender on the Preservability of Cattle Bull Semen

International Journal of Veterinary Science ◽

10.37422/ijvs/034 ◽

2020 ◽

Keyword(s):

Free Radicals ◽

Sperm Motility ◽

Moringa Oleifera ◽

Membrane Integrity ◽

Egg Yolk ◽

Natural Antioxidant ◽

Herbal Supplement ◽

Bull Semen ◽

Before And After ◽

Sperm Membrane

Moringa oleifera extract is a strong natural antioxidant that when was added to the semen extenders, it induced a cryoprotection to spermatozoa effect through elimination of the excess free radicals. So, the existing study intended for clarification of the consequence of extract of Moringa leaves (MLE) on bull spermatozoa after chilling and cryopreservation. MLE concentrations were 0% (control), 10, 20, 30, 40 and 50% (v/v) [MLE: TCF (Tris-citric-fructose diluent)] then 20% egg yolk was added, then the extended semen was assigned to the freezing protocol. Then, it was evaluated for (motility, alive, abnormality %, sperm membrane integrity % before and after freezing). Sperm motility was kept high with the concentration 10, 30 and 40% of MEEY till 8 days of chilling. The concentration 20% maintained sperm motility high till 7 days of chilling. Addition of MLE to TCF significantly (P<0.002) improved sperm motility in all concentrations except the 50% moringa enriched extender with egg yolk (MEEY) where sperm motility was maintained as the control. The use of MEEY maintained % of alive sperms and % of normal spermatozoal membrane (HOST%) as good as the control. In conclusion: moringa as a herbal supplement to semen diluents enhanced preservation in cooled and cryopreserved cattle bull semen.

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